Temporal regulation of expression of immediate early and second phase transcripts by endothelin-1 in cardiomyocytes

Timothy E Cullingford¹ , Thomais Markou¹ , Stephen J Fuller¹ , Alejandro Giraldo¹ , Sampsa Pikkarainen¹ , Georgia Zoumpoulidou¹ , Ali Alsafi¹ , Collins Ekere¹ , Timothy J Kemp¹ , Jayne L Dennis² , Laurence Game² , Peter H Sugden¹ and Angela Clerk¹

¹National Heart and Lung Institute Division, Faculty of Medicine, Imperial College London, Armstrong Road, London SW7 2AZ, UK

²Clinical Sciences Centre/Imperial College Microarray Centre, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK

author email corresponding author email

Genome Biology 2008, 9:R32doi:10.1186/gb-2008-9-2-r32


Published:	14 February 2008

Subject areas: Cell biology, Genome studies

Abstract

Background

Endothelin-1 stimulates Gq protein-coupled receptors to promote proliferation in dividing cells or hypertrophy in terminally differentiated cardiomyocytes. In cardiomyocytes, endothelin-1 rapidly (within minutes) stimulates protein kinase signaling, including extracellular-signal regulated kinases 1/2 (ERK1/2; though not ERK5), with phenotypic/physiological changes developing from approximately 12 h. Hypertrophy is associated with changes in mRNA/protein expression, presumably consequent to protein kinase signaling, but the connections between early, transient signaling events and developed hypertrophy are unknown.

Results

Using microarrays, we defined the early transcriptional responses of neonatal rat cardiomyocytes to endothelin-1 over 4 h, differentiating between immediate early gene (IEG) and second phase RNAs with cycloheximide. IEGs exhibited differential temporal and transient regulation, with expression of second phase RNAs within 1 h. Of transcripts upregulated at 30 minutes encoding established proteins, 28 were inhibited >50% by U0126 (which inhibits ERK1/2/5 signaling), with 9 inhibited 25-50%. Expression of only four transcripts was not inhibited. At 1 h, most RNAs (approximately 67%) were equally changed in total and polysomal RNA with approximately 17% of transcripts increased to a greater extent in polysomes. Thus, changes in expression of most protein-coding RNAs should be reflected in protein synthesis. However, approximately 16% of transcripts were essentially excluded from the polysomes, including some protein-coding mRNAs, presumably inefficiently translated.

Conclusion

The phasic, temporal regulation of early transcriptional responses induced by endothelin-1 in cardiomyocytes indicates that, even in terminally differentiated cells, signals are propagated beyond the primary signaling pathways through transcriptional networks leading to phenotypic changes (that is, hypertrophy). Furthermore, ERK1/2 signaling plays a major role in this response.